Osteoarthritis (OA) is a widespread and increasingly prevalent disorder impairing quality of life for tens of millions of patients. Evidence from study of human OA and several animal OA models, suggests that during OA progression a recapitulation of chondrocyte phenotypic maturation occurs resembling embryonic endochondral ossification. In the prior funding period we identified a number of phenotypic maturational characteristics, which are expressed in OA cartilage, including PTHrP, MMP9, MMP13, Ihh, and BMP6. Articular chondrocytes are extremely refractory in culture to induction of this maturational cascade and are relatively unresponsive to growth factors. However, treatment with azacytidine, which allows demethylation of suppressed genes, induces the normal cascade of chondrocyte maturation, resulting in expression of type X collagen and other hypertrophic markers. Study of the specific signaling pathways involved have implicated a Smad ubiquitinating ligase, Smurf2, as a potential key regulatory step in shifting signal dominance from the TGF-beta to the BMP Smad pathway, leading to chondrocyte maturation. Smurf2 was found to be expressed in human OA but not normal articular cartilage, and is induced by azacytidine treatment in culture. Smurf2 stimulation or over-expression leads to Smad2 and Smad3 degradation, down-regulation of TGF-beta signaling, and up-regulation of BMP signaling. This is sufficient to initiate the entire cascade of chondrocyte maturation and hypertrophy in culture. Specific Aim (1): An investigation of the role of Smurfs in regulation of chondrocyte phenotype in vitro will utilize articular and growth plate chondrocytes in culture to study Smurf1 and 2 effects on Smad signaling and chondrocyte phenotype. The regulation of Smurf expression by relevant growth factors and cytokines will also be studied. Specific Aim (2): Investigation of the role of Smurf2 in chondrocyte maturation and OA in vivo will use a chick limb bud system to analyze effects of Smurf2 on chondrogenesis and maturation during development. A transgenic murine model of cartilage targeted Smurf2 over-expression will be created, and viral transduction of chondrocytes in vivo will be utilized to determine if Smurf2 over-expression can induce, or its inhibition suppress, OA. Finally, the expression of Smurf1 and Smurf2 in human normal and OA cartilage will be examined. These studies should provide a comprehensive picture of the function of Smurfs in regulating normal and OA cartilage phenotype.